CN113563298B - A kind of water-soluble substituent rhodamine fluorescent dye and its preparation method and application - Google Patents

Abstract

The invention discloses rhodamine dyes containing water-soluble substituents and functional derivatives (zinc ion probes and antibody labeling dyes) thereof, which have a structure shown in a general formula I. The biocompatibility of the dye mother nucleus is greatly improved after the N-position of the dye mother nucleus is connected with different water-soluble substituents, and the dye mother nucleus is particularly characterized in the aspects of remarkably reduced non-specific staining in cells, no apoptosis signal, remarkably reduced phototoxicity (zinc ion probe), improved marking efficiency, prolonged observation time (antibody marking dye) and the like. The fluorescent dye can be applied to the fields of observing insulin release of isolated living islets, immunofluorescence labeling and the like.

Description

A kind of water-soluble substituent rhodamine fluorescent dye and its preparation method and application

technical field

The invention relates to a class of rhodamine fluorescent dyes containing water-soluble substituents, in particular to the field of fluorescent dyes in biological fluorescence analysis, especially its application in biological systems.

Background technique

Rhodamine-based fluorescent dyes have longer absorption and emission spectra, higher brightness and better photostability, and have been widely used in biomarkers (proteins and nucleic acids), single-molecule tracers, ion detection and other fields in recent years. It has gradually become an irreplaceable and important tool for life science research. However, previous work often paid more attention to the modification of the photophysical properties of rhodamine dyes, while the modification of improving the biocompatibility of rhodamine dyes was rarely reported. In recent years, the development of advanced imaging technologies such as super-resolution fluorescence microscopy, spinning disk confocal microscopy, total internal reflection microscopy, and light sheet microscopy has put forward new requirements for the development and transformation of fluorescent dyes: on the basis of adjusting the photophysical properties of fluorescent dyes, more Attention should be paid to improving the biocompatibility of dyes to meet the needs of long-term in vivo fluorescence imaging.

Zinc ions (Zn ²⁺ ) play an important role in the physiological and pathological processes in organisms. The vast majority of intracellular zinc ions are tightly bound to proteins, while a small part dissociates in the cell fluid to form free zinc pools. The concentration of zinc ions in different tissues in the organism varies by as much as 8 orders of magnitude. The concentration of free zinc in the cell fluid is about 10 ^-10 M and the concentration in the islet vesicles is as high as 10 ^-2 M. This is due to the interaction between insulin and zinc The ions are stored in the islet vesicles in the form of 4:2 hexamers, and the physiological state of the islets releases insulin and zinc ions into the blood through the exocytosis of secretory vesicles after being stimulated by glucose. Many fluorescent probes have been used for the monitoring of zinc ions, but their general affinity is too high (K _d at the nM level) to be suitable for monitoring the dynamic change process of high-concentration zinc ions in organisms; in addition, silicon probes with far-red emission The synthesis steps of rhodamine-based functional dyes (zinc, calcium ion dyes, etc.) are cumbersome and the yield is low, which greatly limits the development of far-red and near-infrared functional dyes, and cannot be combined with probes with other emission wavelengths (such as The green calcium ion fluorescent probe GCaMP6f) is used to indicate the complex signal transduction process in the tissue; the only low-affinity red-emitting zinc ion probe RhodZin-1 that has been reported in the literature is also monitoring insulin/zinc ion co-expression. During the release process, there is obvious non-specific binding phenomenon in the cell and finally leads to cell apoptosis. This fully demonstrates that the zinc ion fluorescent probe toolbox still needs further development.

Immunofluorescence plays an important role in the detection and identification of various biologically active substances such as proteins, cytokines, cell surface antigens, and tumor markers. According to the principle of antigen-antibody reaction, immunofluorescence covalently couples fluorescent dyes to antigens or antibodies, binds to corresponding antigens or antibodies, and then observes the fluorescent signals through a microscope to determine the location and properties of antigens or antibodies. It is generally believed that fluorescent dyes with better water solubility have higher antibody labeling efficiency. At present, Cy5 sulfonate is the main antibody-labeled dye with far-red emission, but this dye has low labeling efficiency, poor photostability, and obvious non-specific staining, making it difficult to adapt to the requirements of long-term immunofluorescence imaging. Although silicon-substituted rhodamine also has far-red emission wavelength and high molar extinction coefficient, its water solubility and chemical stability are poor, so it has not been widely used in immunofluorescence labeling.

In summary, the problems of poor biocompatibility (high phototoxicity, non-specific labeling, poor water solubility) of current rhodamine dyes and their functionalized derivatives seriously limit their application in biological imaging, but there is still a lack of A general solution to this problem. Therefore, it is of great significance to propose a universal solution to improve the biocompatibility of rhodamine and to construct functional dyes with good biocompatibility based on rhodamine precursors.

Contents of the invention

The purpose of the present invention is to address the problems of poor biocompatibility (high phototoxicity, non-specific labeling, poor water solubility) existing in rhodamine dyes and functionalized derivatives thereof, and propose a A universal solution for linking different water-soluble substituents and applying it to the construction of low-affinity membrane-impermeable zinc ion probes with red and far-red emission spectra and antibody-labeled dyes, and co-expression of insulin and zinc in living islet tissue Fields such as long-term monitoring of release and immunofluorescence labeling have applications.

The present invention firstly provides a class of rhodamine functional dyes substituted by N-water-soluble groups, the general formula of which is:

Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ are independent of each other.

R ₁ is O, C(CH ₃ ) ₂ , Si(CH ₃ ) ₂ , P(O)CH ₃ , PO ₂ ⁻ , PO ₂ CH ₂ CH ₃ or SO ₂ .

R ₂ is O, C, P, S, NH, N ⁺ HCH ₃ , N ⁺ (CH ₃ ) ₂ , P(O)CH ₃ or SO ₂ .

R ₃ is CH ₂ , CHCH ₃ , C(CH ₃ ) ₂ , CHF, CF ₂ , CHCl, CCl ₂ and various possible connections between them.

R ₄ and R ₈ are H, CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , COO ^- , COOMe, CN, SO ₂ CH ₃ , SO ₂ NH ₂ or SO2N(CH3)2.

R ₅ and R ₇ are H, OH, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ OCH ₃ , OCH ₂ COOH, OCH ₂ C ₆ H ₅ N, COOH or the following structures.

Wherein, R ₉ is H, F, Cl, Br, CH ₃ , OCH ₃ or NO ₂ .

R ₆ is H, COOH, N(CH ₂ COOH) ₂ or N(CH ₂ COOCH ₂ OCOCH ₃ ).

X ^- is a halogen ion, ClO ₄ ^- , PF ₆ ^- , BF ₄ ^- , CH ₃ COO ^- and CF ₃ COO ^- .

Preferably, the present invention provides a series of membrane-impermeable fluorescent dyes PK Zinc Red 1-5 with red emission on the basis of general formula I, the structure of which is shown in general formula II; they have lower affinity (K _d from 190 nanomolar to 74 micromolar) and good biocompatibility. The invention also provides a synthesis method of the series of dyes and describes its application in detection and quantification of zinc ion concentration in in vitro samples, long-term monitoring of co-release of insulin and zinc ions on living pancreas tissue, and the like.

The invention provides a preparation method of the above-mentioned dye PK Zinc Red 1-5, which specifically includes the following steps.

Preferably, the present invention provides a series of membrane-impermeable fluorescent dyes PK Zinc Red 1-3 with far-red emission on the basis of general formula I, whose structure is shown in general formula III; they also have lower affinity ( K _d 30 micromolar) and good biocompatibility, not only suitable for long-term monitoring of insulin and zinc ion co-release in living pancreatic islets, but also compatible with blue nuclear dye Hochest, green calcium ion probe GCaMP6f and red mitochondrial dye PK Mito Red is combined for multicolor imaging to explain the complex signal transmission relationship between them.

The present invention provides the preparation method of the above-mentioned dye PK Zinc Red 1-3, compared with the synthetic route reported by the predecessors (J.Am.Chem.Soc.133,36:14157-14159), the preparation method reported herein The reaction conditions are milder and the productive rate is higher, which is more suitable for the gram-level preparation of dyestuffs, and specifically includes the following steps:

The choice of detection methods for zinc ions varies with the properties of fluorescent probes, systems, cells, and tissues. The preferred detection technology solutions are on equipment such as microplate readers, fluorescence spectrophotometers, and spinning disk confocal microscopes. Achieved.

Preferably, the present invention also provides a far-red emitting fluorescent dye PK SiRMorpho on the basis of the general formula I, its structure is shown in IV, and the dye antibody labeling efficiency is higher (on average, each antibody labels 4 dyes) Compared with commercial dyes such as Alexa 647 and Cy5, it shows higher photostability in immunofluorescence imaging.

(IV)

The invention provides the preparation method of above-mentioned dye PK SiR Morpho, specifically comprises the following steps:

When using the above-mentioned dye PK SiR Morpho for immunofluorescence imaging, the preferred detection technology scheme is realized on equipment such as confocal microscope and high-content live cell imaging system.

Description of drawings

Fig. 1 is the fluorescence and absorption spectrum diagrams of the compound PK Zinc Red-1 prepared in the present invention under various zinc ion concentrations.

Fig. 2 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc Red-1 prepared in the present invention.

Fig. 3 is the fluorescence and absorption spectra of the compound PK Zinc Red-2 prepared in the present invention under various zinc ion concentrations.

Fig. 4 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc Red-2 prepared in the present invention.

Fig. 5 is the fluorescence and absorption spectra of the compound PK Zinc Red-3 prepared in the present invention at various zinc ion concentrations.

Fig. 6 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc Red-3 prepared in the present invention.

Fig. 7 is the fluorescence and absorption spectra of the compound PK Zinc Red-4 prepared in the present invention at various zinc ion concentrations.

Fig. 8 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc Red-4 prepared in the present invention.

Fig. 9 is the fluorescence and absorption spectra of the compound PK Zinc Red-5 prepared in the present invention at various zinc ion concentrations.

Fig. 10 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc Red-5 prepared in the present invention.

Fig. 11 is the fluorescence and absorption spectra of the compound PK Zinc FarRed-1 prepared in the present invention at various zinc ion concentrations.

Fig. 12 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc FarRed-1 prepared in the present invention.

Fig. 13 is the fluorescence and absorption spectra of the compound PK Zinc FarRed-2 prepared in the present invention at various zinc ion concentrations.

Fig. 14 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc FarRed-2 prepared in the present invention.

Fig. 15 is the fluorescence and absorption spectra of the compound PK Zinc FarRed-3 prepared in the present invention at various zinc ion concentrations.

Fig. 16 is a data diagram of the zinc ion concentration titration test of the compound PK Zinc FarRed-3 prepared in the present invention.

Figure 17 is a comparison chart of the experimental results of the compound PK Zinc Red-1 prepared by the present invention and the reported compound RhodZin-1 on the complete isolated living pancreatic islets of mice for insulin/zinc ion co-release detection; wherein, in Figure A The signal in ROI1 is the apoptosis signal, the signal in ROI 2 is the intracellular non-specific labeling signal, ROI 3 in Figure B is the intracellular background: the fluorescent spot in ROI 4 is the co-release signal of insulin/zinc ions.

Figure 18 is an experimental result diagram of the compound PK Zinc Red-5 prepared by the present invention on the mouse islet cell mass for insulin/zinc ion co-release detection; the dotted line shows the outer contour of the cell mass, and the fluorescent point in the figure is PK ZincRed -5 detection of insulin/zinc ion co-release signal;

Figure 19 is a graph showing the experimental results of the detection of insulin/zinc ion co-release on the complete isolated live pancreatic islets of mice under the stimulation of the drug forskolin by the compound PK Zinc Red-1 prepared by the present invention; where the fluorescent dye FM 4- 64 marked the cell membrane, and the fluorescent spots in the figure are the co-release signals of insulin/zinc ions detected by PK Zinc Red-1

Fig. 20 is a graph showing the experimental results of multicolor imaging of the compound PK Zinc Far Red-3 prepared in the present invention combined with the blue nuclear dye Hochest, the green calcium ion probe GCaMP6f and the red mitochondrial dye PK Mito Red;

Fig. 21 is a graph showing the experimental results of fluorescence signal changes when the compound PK SiR Morpho prepared in the present invention is used for immunofluorescence imaging with commercial dyes Alexa 647 and Cy5.

Detailed ways:

The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

The synthesis method of the N-water-soluble group-substituted rhodamine fluorescent probe of the present invention will be further described below in conjunction with examples.

Example 1: the synthesis of compound PK Zinc Red-1:

(1) Synthesis of compound 1b:

First, compound 1a (1.00 g, 3.39 mmol, 1.0 eq) was dissolved in 10 mL of dry DMF and stirred at -20°C for 30 min. Then, POCl ₃ (2.88g, 18.8mmol, 5.5eq) was added dropwise into the above solution, and the temperature was raised to 60°C for 3h. After the above reaction was completed, the resulting solution was slowly dripped into 20 mL of ice-water mixed liquid, then the reaction liquid was neutralized with Na ₂ CO ₃ solution and extracted three times with dichloromethane (40 mL each time), the organic layer was collected and washed with Na ₂ SO ₄ The solid dried. The resulting solution was concentrated under vacuum/reduced pressure, and purified by silica gel flash column chromatography (developing solvent 10% ethyl acetate/petroleum ether) to obtain yellow solid compound 1b (920 mg, 2.84 mmol, yield 84%).

¹ H NMR (400MHz, Chloroform-d) δ9.78(s, 1H), 7.36–7.32(m, 2H), 6.74(d, J=8.6Hz, 1H), 4.23(q, J=7.2Hz, 4H ), 4.18 (s, 4H), 3.83 (s, 3H), 1.29 (t, J=7.1Hz, 6H).

¹³ C NMR (101 MHz, Chloroform-d) δ190.79, 170.96, 150.67, 145.11, 130.10, 126.79, 116.46, 110.27, 61.18, 55.89, 54.43, 14.42.

(ESI) calcd for _C16H22NO6 ⁺ [ _M +H] ⁺ 324.14, found 324.35 _.

(2) Synthesis of compound 1c:

At room temperature, 3-morpholinephenol (346mg, 1.94mmol, 2.5eq) and p-toluenesulfonic acid (13.3mg, 0.0772 mmol, 0.1eq), followed by stirring at 80°C for 12h. After the reaction was completed, the resulting mixture was dissolved in 50 mL CHCl ₃ , and then washed three times with 3M NaOAc in water. The organic layers were collected and concentrated under vacuum/reduced pressure. Then the crude product was dissolved in a 1:1 DCM/MeOH mixture (4mL/4mL), DDQ (total amount 176mg, 0.775mmol, 1.0eq) was added three times, and the reaction was continued at room temperature for 4h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) afforded the trifluoroacetate salt of compound 1c (80 mg, 0.12 mmol, yield 16%) as a purple-black solid .

¹ H NMR (400MHz, Methanol-d ₄ )δ7.67(d, J=9.6Hz, 2H), 7.30(dd, J=9.6, 2.6Hz, 2H), 7.18(d, J=2.5Hz, 2H) ,7.09(d,J=1.9Hz,1H),7.03(dd,J=8.2,1.9Hz,1H),6.96(d,J=8.2Hz,1H),4.29–4.21(m,8H),3.88– 3.83 (m, 8H), 3.81 (s, 3H), 3.78–3.73 (m, 8H), 1.32 (t, J=7.1Hz, 6H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ173.04, 160.37, 159.96, 158.73, 151.77, 142.88, 133.64, 130.85, 125.16, 124.85, 118.27, 115.81, 115.58, 98.55, 67 .45, 62.10, 56.62, 55.42, 48.36, 14.61 .

HRMS (ESI) calculated value C ₃₆ H ₄₂ N ₃ O ₈ ⁺ [M] ⁺ 644.2966, actual value 644.2986.

(3) Synthesis of compound PK Zinc Red-1:

To compound 1c (50 mg, 77 μmol, 1.0 eq) in MeOH/H ₂ O (4 mL/1 mL) was added 2M LiOH aqueous solution (388 μL, 10.0 eq), and the resulting mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 35% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave the trifluoroacetate salt of PK Zinc Red-1 as a purple-black solid compound (15 mg, 26 μmol, yield 34%).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.68(d, J=9.6Hz, 2H), 7.29(dd, J=9.6, 2.3Hz, 2H), 7.18(d, J=2.5Hz, 2H) ,7.09(d,J＝1.9Hz,1H),7.04–6.97(m,2H),4.24(s,4H),3.87–3.85(m,8H),3.82(s,3H),3.76–3.73(m ,8H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ175.04, 160.41, 159.96, 158.72, 151.71, 142.95, 133.69, 130.85, 125.03, 124.92, 118.03, 115.79, 115.57, 98.55, 67 .45, 56.54, 55.45, 48.36.

HRMS (ESI) calculated value C ₃₂ H ₃₆ N ₃ O ₈ ⁺ [M] ⁺ 588.2340, actual value 588.2362.

Example 2: Synthesis of Compound PK Zinc Red-2:

(1) Synthesis of compound 2b:

Firstly, compound 2a (730 mg, 2.36 mmol, 1.0 eq) was dissolved in 10 mL of dry DMF and stirred at -20°C for 30 min. Then, POCl ₃ (3.00 g, 19.6 mmol, 8.3 eq) was added dropwise into the above solution, and the resulting mixture was heated to 60° C. for 3 h. After the above reaction was completed, the resulting solution was slowly dripped into 20 mL of ice-water mixture, then the mixture was neutralized with Na ₂ CO ₃ solution and extracted three times with dichloromethane (40 mL each time), the organic layer was collected and washed with Na ₂ SO ₄ solids dry. The resulting solution was concentrated under vacuum/reduced pressure, and purified by silica gel flash column chromatography (developing solvent 10% ethyl acetate/petroleum ether) to obtain compound 2b (680 mg, 2.02 mmol, yield 86%) as a yellow solid.

¹ H NMR (400MHz, Chloroform-d) δ9.77(s,1H),7.35–7.31(m,2H),6.71(d,J=8.5Hz,1H),4.28–4.17(m,8H),4.07 (q, J=6.9Hz, 2H), 1.38(t, J=6.9Hz, 3H), 1.30(t, J=7.1Hz, 6H).

¹³ C NMR (101 MHz, Chloroform-d) δ190.79, 171.02, 149.89, 145.20, 129.99, 126.57, 116.23, 110.76, 64.42, 61.16, 54.07, 14.50, 14.34.

MS (ESI) calcd for C ₁₇ H ₂₄ NO ₆ ⁺ [M+H] ⁺ 338.16, found 338.21.

(2) Synthesis of compound 2c:

At room temperature, 3-morpholinephenol (66.0mg, 0.371mmol, 2.5eq) and p-toluenesulfonic acid (2.6mg, 14.8μmol, 0.1eq), then stirred at 80°C for 12h. After the reaction was complete, the resulting mixture was dissolved in 25 mL CHCl ₃ , and then washed with 3M NaOAc in water. The organic layers were collected and concentrated under vacuum/reduced pressure. The crude product was then dissolved in a 1:1 DCM/MeOH mixture (2 mL/2 mL), and DDQ (total amount 34.0 mg, 0.148 mmol, 1.0 eq) was added three times, and the reaction was continued at room temperature for 4 h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give the trifluoroacetate salt of compound 2c (11 mg, 17 μmol, yield 11%) as a purple-black solid.

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.66 (dd, J=9.5, 2.3Hz, 2H), 7.29 (d, J=9.4Hz, 2H), 7.17 (d, J=2.1Hz, 2H) ,7.05(d,J=1.6Hz,1H),7.02–6.92(m,2H),4.30–4.20(m,8H),4.04(q,J=6.9Hz,2H),3.89–3.81(m,8H ),3.77–3.71(m,8H),1.40–1.29(m,9H). ¹³ C NMR(101MHz,Methanol-d ₄ )δ173.04,160.35,159.91,158.69,150.97,142.97,133.63,125.12,124.72,1 18.26 , 116.25, 115.81, 115.53, 98.59, 67.45, 65.91, 62.13, 55.12, 48.39, 14.97, 14.55.

HRMS (ESI) calculated value C ₃₇ H ₄₄ N ₃ O ₈ ⁺ [M] ⁺ 658.3123, actual value 658.3134.

(3) Synthesis of compound PK Zinc Red-2:

To compound 2c (10.0mg, 15.2μmol, 1.0eq) MeOH/H ₂ O (2mL/0.5mL) mixed solution was added 2M LiOH aqueous solution (78μL, 10.0eq), and the resulting mixture was stirred at room temperature for 2h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave the trifluoroacetate salt of PK Zinc Red-2 as a purple-black solid compound (2.1 mg, 3.3 μmol, yield 22%).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.66 (d, J=9.5Hz, 2H), 7.28 (dd, J=9.6, 2.1Hz, 2H), 7.17 (d, J=2.2Hz, 2H) ,7.05(d,J=1.6Hz,1H),7.02–6.93(m,2H),4.24(s,4H),4.05(q,J=6.9Hz,2H),3.88–3.84(m,8H), 3.76–3.73 (m, 8H), 1.42 (t, J=6.9Hz, 3H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ175.10, 160.36, 159.90, 158.67, 150.95, 143.07, 133.66, 124.95, 124.77, 117.95, 116.24, 115.78, 115.50, 98.58, 67 .45, 66.07, 55.25, 48.38, 14.86.

HRMS (ESI) theoretical value C ₃₃ H ₃₆ N ₃ O ₈ ⁺ [M] ⁺ 602.2497, actual value 602.2488.

Example 3: Synthesis of Compound PK Zinc Red-3:

(1) Synthesis of compound 3b:

Firstly, compound 3a (700 mg, 2.06 mmol, 1.0 eq) was dissolved in 10 mL of dry DMF and stirred at -20°C for 30 min. Then, POCl ₃ (2.52 g, 16.5 mmol, 8.0 eq) was added dropwise into the above solution, and the resulting mixture was heated to 60° C. for 3 h. After the above reaction was completed, the resulting solution was slowly dripped into 20 mL of ice-water mixture, then the mixture was neutralized with Na ₂ CO ₃ solution and extracted three times with dichloromethane (40 mL each time), the organic layer was collected and washed with Na ₂ SO ₄ solids dry. The resulting solution was concentrated under vacuum/reduced pressure, and purified by silica gel flash column chromatography (developing solvent 10% EtOAc/hexane) to obtain compound 3b (500 mg, 1.36 mmol, yield 66%) as a yellow oil.

¹ H NMR (400MHz, Chloroform-d) δ9.76(s,1H),7.36–7.34(m,2H),6.73(d,J=8.6Hz,1H),4.27–4.19(m,8H),4.17 -4.14 (m, 2H), 3.71 - 3.67 (m, 2H), 3.39 (s, 3H), 1.29 (t, J=7.2Hz, 6H).

¹³ C NMR (101 MHz, Chloroform-d) δ190.70, 170.86, 149.77, 145.23, 129.95, 126.79, 116.67, 111.64, 70.61, 68.07, 61.15, 58.94, 54.00, 14.34.

MS (ESI) theoretical value C ₁₈ H ₂₆ NO ₇ ⁺ [M+H] ⁺ 368.17, actual value 368.58.

(2) Synthesis of compound 3c:

At room temperature, 3-morpholinephenol (61.0mg, 0.340mmol, 2.5eq) and p-toluenesulfonic acid (2.3mg, 13.6μmol, 0.1eq), then stirred at 80°C for 12h. After the reaction was complete, the resulting mixture was dissolved in 20 mL CHCl ₃ , and then washed with 3M NaOAc in water. The organic layers were collected and concentrated under vacuum/reduced pressure. Then the crude product was dissolved in a 1:1 DCM/MeOH mixture (2 mL/2 mL), and DDQ (total amount 31.0 mg, 0.136 mmol, 1.0 eq) was added three times, and the reaction was continued at room temperature for 4 h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give the trifluoroacetate salt of purple-black solid compound 3c (5.0 mg, 7.2 μmol, yield 5% ).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.65 (d, J=9.4Hz, 2H), 7.28 (dd, J=9.7, 2.2Hz, 2H), 7.15 (d, J=2.2Hz, 2H) ,7.12(d,J=1.7Hz,1H),7.06–6.93(m,2H),4.31(s,4H),4.25(q,J=7.1Hz,4H),4.16–4.12(m,2H), 3.87–3.84(m,8H), 3.76–3.72(m,8H), 3.70–3.68(m,2H), 3.39(s,3H), 1.32(t,J=7.1Hz,6H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ172.85, 160.17, 159.86, 158.67, 151.01, 142.97, 133.64, 125.13, 125.11, 118.70, 117.08, 115.82, 115.49, 98.58, 72 .00, 69.77, 67.45, 62.13, 59.19, 55.00 , 48.39, 14.57.

HRMS (ESI) theoretical value C ₃₈ H ₄₆ N ₃ O ₉ ⁺ [M] ⁺ 688.3229, actual value 688.3262.

(3) Synthesis of compound PK Zinc Red-3:

To compound 3c (5.0mg, 7.25μmol, 1.0eq) MeOH/H ₂ O (1.2mL/0.3mL) mixed solution was added 2M LiOH aqueous solution (40μL, 10.0eq), and the resulting mixture was stirred at room temperature for 2h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave the trifluoroacetate salt of PK Zinc Red-3 as a purple-black solid compound (2.0 mg, 3.1 μmol, yield 44%).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.64 (d, J=9.5Hz, 2H), 7.26 (dd, J=9.5, 2.2Hz, 2H), 7.13 (d, J=2.2Hz, 2H) ,7.09(d,J＝1.7Hz,1H),7.03–6.96(m,2H),4.27(s,4H),4.16–4.11(m,2H),3.92–3.82(m,10H),3.74–3.71 (m,8H),3.40(s,3H).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ174.88, 160.19, 159.86, 158.65, 150.92, 143.04, 133.65, 125.13, 124.98, 118.28, 116.5, 115.76, 115.46, 98.56, 71. 96, 69.83, 67.43, 59.23, 55.01, 48.36 .

HRMS (ESI) theoretical value C ₃₄ H ₃₈ N ₃ O ₉ ⁺ [M] ⁺ 632.2603, calculated value 632.2616.

Example 4: Synthesis of Compound PK Zinc Red-4:

(1) Synthesis of compound 4b:

Firstly, compound 4a (800 mg, 2.18 mmol, 1.0 eq) was dissolved in 10 mL of dry DMF and stirred at -20°C for 30 min. Then, POCl ₃ (2.66g, 17.4mmol, 8.0eq) was added dropwise into the above solution, and the resulting mixture was heated to 60°C for 3h. After the above reaction was completed, the resulting solution was slowly dripped into 20 mL of ice-water mixture, then the mixture was neutralized with Na ₂ CO ₃ solution and extracted three times with dichloromethane (40 mL each time), the organic layer was collected and washed with Na ₂ SO ₄ solids dry. The resulting solution was concentrated under vacuum/reduced pressure, and purified by silica gel flash column chromatography (developing solvent 10% ethyl acetate/petroleum ether) to obtain yellow solid compound 4b (518mg, 1.31mmol, yield 60%).

¹ H NMR (400MHz, Chloroform-d) δ9.76(s, 1H), 7.40(dd, J＝8.2, 1.8Hz, 1H), 7.26(d, J＝1.6Hz, 1H), 6.81(d, J =8.2Hz, 1H), 4.65(s, 2H), 4.31–4.17(m, 10H), 1.33–1.23(m, 9H).

¹³ C NMR (101MHz, Chloroform-d) δ190.33, 170.67, 168.06, 162.54, 148.66, 145.29, 129.79, 127.23, 117.17, 111.73, 65.62, 61.41, 61.04, 54.07, 36.46, 31 .39, 14.21, 14.11.

MS (ESI) calcd for C ₁₉ H ₂₇ NO ₈ ⁺ [M+H] ⁺ 396.17, calcd. 396.26.

(2) Synthesis of compound 4c:

At room temperature, 3-morpholinephenol (57.0mg, 0.316mmol, 2.5eq) and p-toluenesulfonic acid (2.2mg, 12.7μmol, 0.1eq), then stirred at 80°C for 12h. After the reaction was complete, the resulting mixture was dissolved in 20 mL CHCl ₃ , and then washed with 3M NaOAc in water. The organic layers were collected and concentrated under vacuum/reduced pressure. Then the crude product was dissolved in a 1:1 DCM/MeOH mixture (2 mL/2 mL), and DDQ (29.0 mg, 0.127 mmol, 1.0 eq in total) was added three times, and the reaction was continued at room temperature for 4 h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give the trifluoroacetate salt of compound 4c (5.2 mg, 7.3 μmol, yield 6% ).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.60(d, J=9.6Hz, 2H), 7.26(dd, J=9.6, 2.3Hz, 2H), 7.14(d, J=2.4Hz, 2H) ,7.11–7.03(m,3H),4.75(s,2H),4.33(s,4H),4.28–4.17(m,6H),3.89–3.82(m,8H),3.76–3.70(m,8H) , 1.29 (t, J=7.1Hz, 6H), 1.23 (t, J=7.1Hz, 3H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ172.77, 170.19, 159.66, 159.49, 158.54, 149.85, 143.20, 133.58, 130.80, 126.12, 124.95, 119.28, 115.92, 115.30, 9 8.79, 67.52, 62.54, 62.16, 55.13, 53.31 , 48.57, 14.63, 14.43.

HRMS (ESI) theoretical value C ₃₉ H ₄₆ N ₃ O ₁₀ ⁺ [M] ⁺ 716.3178, calculated value 716.3196.

(3) Synthesis of compound PK Zinc Red-4:

To compound 4c (5.2mg, 7.3μmol, 1.0eq) mixed solution of MeOH/H ₂ O (1.2mL/0.3mL) was added 2M LiOH aqueous solution (40μL, 10.0eq), and the resulting mixture was stirred at room temperature for 2h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave the trifluoroacetate salt of PK Zinc Red-4 as a purple-black solid compound (2.5 mg, 4.0 μmol, yield 55%).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.64 (d, J=9.6Hz, 2H), 7.25 (dd, J=9.6, 2.5Hz, 2H), 7.13 (d, J=2.4Hz, 2H) ,7.09–7.02(m,3H),4.75(s,2H),4.32(s,4H),3.87–3.83(m,8H),3.76–3.71(m,8H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ174.90, 172.07, 159.87, 159.78, 158.63, 150.01, 143.29, 133.62, 130.85, 125.85, 124.98, 119.02, 115.76, 115.37, 9 8.57, 67.43, 66.43, 55.11, 48.36.

HRMS (ESI) theoretical value C ₃₃ H ₃₄ N ₃ O ₁₀ ⁺ [M] ⁺ 632.2239, calculated value 632.2235.

Example 5: Synthesis of Compound PK Zinc Red-5:

(1) Synthesis of compound 5c:

Compound 5b was synthesized according to the reported procedure (Org. Lett., 2011, 13(17): 4558-4561). At room temperature, 3-morpholinephenol (99.0mg, 0.55mmol, 2.2eq) and p-toluenesulfonic acid (4.3mg, 25μmol, 0.1eq), then stirred at 80°C for 12h. After the reaction was complete, the resulting mixture was dissolved in 20 mL CHCl ₃ , and then washed with 3M NaOAc in water. The organic layers were collected and concentrated under vacuum/reduced pressure. Then the crude product was dissolved in a 1:1 DCM/MeOH mixture (2 mL/2 mL), and DDQ (57.0 mg, 0.25 mmol, 1.0 eq in total) was added three times, and the reaction was continued at room temperature for 4 h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give the trifluoroacetate salt of compound 5c (24.0 mg, 33 μmol, yield 13%) as a purple-black solid .

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.81–8.77 (m, 1H), 8.39 (td, J=7.8, 1.6Hz, 1H), 7.99 (d, J=8.0Hz, 1H), 7.87– 7.81(m,1H),7.52(d,J=9.6Hz,2H),7.27–7.21(m,3H),7.18–7.12(m,4H),5.48(s,2H),4.31(s,4H) , 4.13 (q, J=7.1Hz, 4H), 3.89–3.83 (m, 8H), 3.77–3.71 (m, 8H), 1.25 (t, J=7.1Hz, 6H).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ172.74, 159.88, 159.43, 158.71, 154.59, 150.46, 145.92, 144.85, 143.66, 133.36, 130.84, 126.72, 126.34, 126.17, 1 20.18, 118.91, 115.88, 115.47, 98.59, 70.44 ,67.43,62.20,55.19,48.40,14.50.

HRMS (ESI) theoretical value C ₄₁ H ₄₅ N ₄ O ₈ ⁺ [M] ⁺ 721.3232, actual value 721.3212.

(2) Synthesis of compound PK Zinc Red-5:

To a mixed solution of compound 5c (24.0 mg, 33 μmol, 1.0 eq) in MeOH/H ₂ O (4 mL/1 mL) was added 2M LiOH aqueous solution (168 μL, 10.0 eq), and the resulting mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 545nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave the trifluoroacetate salt of PK Zinc Red-5 as a purple-black solid compound (10.4 mg, 15.6 μmol, yield 48%).

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.77–8.70 (m, 1H), 8.33 (td, J=7.8, 1.5Hz, 1H), 7.96 (d, J=7.9Hz, 1H), 7.79 ( dd,J=7.6,5.4Hz,1H),7.54(d,J=9.4Hz,2H),7.27–7.20(m,3H),7.19–7.10(m,4H),5.45(s,2H),4.28 (s,4H), 3.88–3.84(m,8H), 3.78–3.73(m,8H).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ174.64, 159.92, 159.58, 158.71, 154.65, 150.27, 146.24, 144.36, 143.73, 133.44, 130.85, 126.55, 126.30, 126.08, 1 19.64, 118.66, 115.84, 115.48, 98.59, 70.63 , 67.44, 55.17, 48.40.

HRMS (ESI) theoretical value C ₃₇ H ₃₇ N ₄ O ₈ ⁺ [M] ⁺ 665.2606, calculated value 665.2606.

Example 6: Synthesis of Compound PK Zinc FarRed-1:

(1) Synthesis of compound two (2-bromo-4-morpholine phenyl) methane:

37% formalin (0.475mL, 6.34mmol, 1.0eq) was added to a solution of 4-(3-bromophenyl)morpholine (3.08g, 12.7mmol, 2.0eq) in AcOH (30mL), followed by The reaction solution was warmed up to 90°C and stirred for 3h. After the reaction was completed and the reaction solution was cooled to room temperature, the reaction solution was neutralized with saturated NaHCO ₃ aqueous solution and the organic layer was extracted with CH ₂ Cl ₂ . The organic layer was washed with brine, dried over _Na2SO4 , and the solvent was _evaporated to dryness. The resulting product was purified by column chromatography (5% EtOAc/hexane) to finally give bis(2-bromo-4-morpholinephenyl)methane (1.36 g, 2.74 mmol, 43% yield) as a white solid.

¹ H NMR (400MHz, Chloroform-d) δ7.12(d, J=2.6Hz, 2H), 6.88(d, J=8.6Hz, 2H), 6.76(dd, J=8.5, 2.6Hz, 2H), 4.03 (s, 2H), 3.86–3.80 (m, 8H), 3.16–3.08 (m, 8H).

¹³ C NMR (101 MHz, Chloroform-d) δ 150.84, 130.97, 130.42, 125.67, 119.59, 114.90, 66.88, 49.15, 40.31.

HRMS (ESI) theoretical value C ₂₁ H ₂₅ Br ₂ N ₂ O ₂ ⁺ [M+H] ⁺ 497.0264, actual value 497.0234.

(2) Synthesis of compound morpholino-siloxanthone:

Add bis(2-bromo-4-morpholinephenyl)methane (600mg, 1.22mmol, 1.0eq) and anhydrous THF (9mL) into a heat-dried flask filled with argon. After the solution was cooled to -78°C, n-BuLi (1.6M n-hexane solution, 1.70 mL, 2.72 mmol, 2.2 eq) was rapidly dropped into n-BuLi with a syringe, and the reaction was stirred at -78°C for 10 min. At the same temperature, SiMe ₂ Cl ₂ (0.165mL, 9.35mmol, 1.2eq) was slowly dropped into it with a syringe, then the reaction solution was raised to room temperature and stirred for 30min. After the reaction was completed, 2M hydrochloric acid was added to quench the reaction, and the reaction solution was neutralized with saturated NaHCO ₃ aqueous solution, and the organic layer was extracted with CH ₂ Cl ₂ . The organic layer was washed with brine, dried over _Na2SO4 , and the solvent was _evaporated to dryness. The obtained crude product (silicon-pyronine) was directly used in the next reaction without post-treatment. The product was dissolved _in 15 mL _CH3COCH3 , and the solution was cooled to 0 °C. Grind KMnO ₄ (578mg, 3.66mmol, 3.0eq) into a fine powder, and put it into the reaction solution several times within 1 hour under stirring. After continuing to react for 1 h at room temperature, add 20 mL of CH ₂ Cl ₂ to dilute the reaction solution, filter through filter paper and evaporate the solvent to dryness (silica gel powder can be added during the process to absorb fine and dense MnO ₂ powder, which is convenient for filtration). The obtained product was purified by column chromatography (10% ethyl acetate/petroleum ether) to obtain a light yellow solid morpholinosiloxanthone (114mg, 0.278mmol, yield 23%).

¹ H NMR (400MHz, Chloroform-d) δ8.39 (d, J=8.7Hz, 2H), 7.06–6.99 (m, 4H), 3.92–3.85 (m, 8H), 3.38–3.32 (m, 8H) ,0.47(s,6H).

¹³ C NMR (101 MHz, Chloroform-d) δ 185.47, 152.62, 140.65, 132.42, 131.76, 117.21, 115.92, 66.82, 47.79, -0.99.

HRMS (ESI) theoretical value C ₂₃ H ₂₉ N ₂ O ₃ Si ⁺ [M+H] ⁺ 409.1947, actual value 409.1916.

(3) Synthesis of compound 1e:

Compound 1d (1.00g, 4.95mmol, 1.0eq), triethylamine (1.45mL, 10.4mmol, 10.0eq) and 4-(dimethylamino)pyridine (60.0mg, 0.50mmol, 0.1eq) were dissolved at room temperature In 20mL CH ₂ Cl ₂ , then slowly drop 5mL of 1,2-bis(chlorodimethylsilyl)ethane (1.17g, 5.45mmol, 1.1eq) in CH ₂ Cl ₂ into the above mixture , stirred for 3h. After the reaction, 30 mL of n-hexane was added to precipitate triethylamine chloride salt, which was then filtered off. The obtained light yellow oil was purified by column chromatography (alumina substrate, 5% ethyl acetate/petroleum ether) to finally obtain compound 1e as a colorless oil (1.58 g, 4.60 mmol, yield 93%).

¹ H NMR (400MHz, Chloroform-d) δ6.95–6.90 (m, 2H), 6.75 (d, J=8.8Hz, 1H), 3.73 (s, 3H), 0.84 (s, 4H), 0.05 (s ,12H).

¹³ C NMR (101 MHz, Chloroform-d) δ 156.31, 134.83, 129.33, 123.44, 115.08, 114.44, 55.00, 8.84, 0.10.

(4) Synthesis of compound 1f:

Add compound 1e (100mg, 0.292mmol, 5.0eq) and anhydrous THF (3mL) in a heat-dried flask filled with argon. After the solution was cooled to -20°C, n-BuLi (1.6M n-hexane solution 0.20mL, 0.32mmol, 5.5eq) was rapidly dropped into n-BuLi with a syringe, and the reaction was stirred at -20°C for 15min. At the same temperature, 1 mL of a solution of siloxanthone (25.0 mg, 57.8 μmol, 1.0 eq) in anhydrous THF was slowly dripped into it with a syringe, and the stirring was continued for 20 min. After the reaction was completed, 2M hydrochloric acid was added to quench the reaction and warmed to room temperature. After neutralizing the reaction solution with saturated NaHCO ₃ aqueous solution, the organic layer was extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , then the reaction solution was concentrated under vacuum/reduced pressure, and purified by HPLC (eluent, 20min linear gradient, solvent B component increased from 30% to 95%; Flow rate 5.0mL/min; detection wavelength 650nm; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to obtain dark blue solid compound 1f after trifluoroacetic acid Salt (16.3 mg, 31.7 μmol, 55% yield).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.53(d, J=2.9Hz, 2H), 7.32(d, J=7.9Hz, 1H), 7.29(d, J=9.7Hz, 2H), 7.06 (d,J=1.7Hz,1H),6.96(dd,J=9.7,2.9Hz,2H),6.89(dd,J=7.9,1.7Hz,1H),3.92(s,3H),3.85–3.82( m,16H), 0.60(s,6H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ170.39, 155.28, 151.78, 150.22, 143.81, 137.61, 131.76, 129.90, 123.90, 122.44, 121.14, 115.32, 114.42, 67.59, 56 .86, 48.36, -1.33.

HRMS (ESI) theoretical value C ₃₀ H ₃₆ N ₃ O ₃ Si ⁺ [M] ⁺ 514.2520, calculated value 514.2495.

(5) Synthesis of compound 1g:

At room temperature, K ₂ CO ₃ (40.0mg, 0.29mmol, 10.0eq), KI (2.4mg, 14μmol, 0.5eq) and bromoacetic acid were added to 1mL of compound 1f (15.0mg, 29μmol, 1.0eq) in DMF Ethyl ester (48mg, 0.29mmol, 10.0eq), the reaction was stirred at 95°C for 12h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 650nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give dark blue solid compound 1g trifluoroacetate (14.3 mg, 20.8 μmol, yield 72% ).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.51(d, J=2.8Hz, 2H), 7.36(d, J=9.7Hz, 2H), 6.94(dd, J=9.7, 2.9Hz, 2H) ,6.89–6.84(m,2H),6.75(dd,J＝8.2,1.9Hz,1H),4.28–4.15(m,8H),3.87–3.76(m,16H),3.75(s,3H),1.30 (t, J = 7.1 Hz, 6H), 0.59 (s, 6H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ173.10, 171.82, 155.22, 151.41, 150.15, 144.15, 141.11, 132.81, 130.20, 123.97, 122.25, 117.99, 115.44, 115.18, 6 7.55, 61.98, 56.52, 55.31, 48.29, 14.62 ,-1.30.

HRMS (ESI) theoretical value C ₃₈ H ₄₈ N ₃ O ₇ Si ⁺ [M] ⁺ 686.3256, calculated value 686.3239.

(5) Synthesis of compound PK Zinc FarRed-1:

To compound 1g (14mg, 20μmol, 1.0eq) MeOH/H ₂ O (2mL/0.5mL) mixed solution was added 2M LiOH aqueous solution (100μL, 10.0eq), and the resulting mixture was stirred at room temperature for 2h. After the reaction was completed, the reaction solution was acidified with 2M hydrochloric acid solution, and then concentrated under vacuum/reduced pressure. The resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 650nm; eluent A: containing 0.1% (v/v) TFA in ddH ₂ O; eluent B: CH ₃ CN) gave dark blue solid compound PK Zinc FarRed-1 as trifluoroacetate salt (8.2 mg, 13 μmol, yield 63%).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.51(d, J=2.9Hz, 2H), 7.37(d, J=9.6Hz, 2H), 6.95(dd, J=9.7, 2.9Hz, 2H) ,6.90(d,J=8.2Hz,1H),6.86(d,J=1.9Hz,1H),6.76(dd,J=8.2,1.9Hz,1H),4.19(s,4H),3.85–3.80( m,16H), 3.77(s,3H), 0.58(s,6H).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ175.28, 171.99, 155.26, 151.39, 150.15, 144.26, 141.16, 132.76, 130.24, 124.00, 122.24, 117.74, 115.45, 115.20, 6 7.59, 56.40, 55.41, 48.30, -1.31.

HRMS (ESI) theoretical value C ₃₄ H ₄₀ N ₃ O ₇ Si ⁺ [M] ⁺ 630.2630, actual value 630.2615.

Example 7: Synthesis of Compound PK Zinc FarRed-2:

(1) Synthesis of compound 2e:

Compound 2d (1.06g, 4.95mmol, 1.0eq), triethylamine (1.45mL, 10.4mmol, 10.0eq) and 4-(dimethylamino)pyridine (60.0mg, 0.50mmol, 0.1eq) were dissolved at room temperature In 20mL CH ₂ Cl ₂ , then slowly drop 5mL of 1,2-bis(chlorodimethylsilyl)ethane (1.17g, 5.45mmol, 1.1eq) in CH ₂ Cl ₂ into the above mixture , stirred for 3h. After the reaction, 30 mL of n-hexane was added to precipitate triethylamine chloride salt, which was then filtered off. The obtained goose-yellow oil was purified by column chromatography (alumina substrate, 5% ethyl acetate/petroleum ether) to finally obtain compound 2e (1.27 g, 3.57 mmol, yield 72%) as a colorless oil.

¹ H NMR (400MHz, Chloroform-d) δ6.95(s,1H),6.75(s,1H),3.70(s,3H),2.27(s,3H),0.84(s,4H),0.06(s ,12H).

¹³ C NMR (101 MHz, Chloroform-d) δ 154.24, 134.76, 130.10, 129.24, 117.05, 114.88, 55.11, 22.11, 8.87, 0.14.

(2) Synthesis of compound 2f:

Compound 2e (100mg, 0.280mmol, 4.8eq) and anhydrous THF (3mL) were added into a heat-dried flask filled with argon. After the solution was cooled to -20°C, n-BuLi (1.6M n-hexane solution 0.19mL, 0.31mmol, 5.3eq) was rapidly dropped into n-BuLi with a syringe, and the reaction was stirred at -20°C for 15min. At the same temperature, 1 mL of a solution of siloxanthone (25.0 mg, 57.8 μmol, 1.0 eq) in anhydrous THF was slowly dripped into it with a syringe, and the stirring was continued for 20 min. After the reaction was completed, 2M hydrochloric acid was added to quench the reaction and warmed to room temperature. After neutralizing the reaction solution with saturated NaHCO ₃ aqueous solution, the organic layer was extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , then the reaction solution was concentrated under vacuum/reduced pressure, and purified by HPLC (eluent, 20min linear gradient, solvent B component increased from 30% to 95%; Flow rate 5.0mL/min; detection wavelength 650nm; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give dark blue solid compound 2f trifluoroacetic acid Salt (19.0 mg, 36.0 μmol, 62% yield).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.56(d, J=2.9Hz, 2H), 7.37(s, 1H), 7.15(d, J=9.6Hz, 2H), 7.02(s, 1H) ,6.96(dd,J＝9.7,2.8Hz,2H),3.91(s,3H),3.89–3.80(m,16H),2.00(s,3H),0.62(s,3H),0.61(s,3H ).

¹³ C NMR (101MHz, Methanol-d ₄ )δ168.80, 155.38, 151.58, 150.19, 142.65, 140.19, 129.95, 129.14, 125.71, 123.14, 122.63, 115.79, 114.15, 67.58, 57 .05, 48.46, 18.52, -1.34, -1.49 .

HRMS (ESI) theoretical value C ₃₁ H ₃₈ N ₃ O ₃ Si ⁺ [M] ⁺ 528.2677, actual value 528.2645.

(3) Synthesis of compound 2g:

At room temperature, K ₂ CO ₃ (38.0mg, 0.28mmol, 10.0eq), KI (2.4mg, 14μmol, 0.5eq) and bromoacetic acid were added to 1mL of compound 2f (15.0mg, 28μmol, 1.0eq) in DMF Ethyl ester (46mg, 0.28mmol, 10.0eq), the reaction was stirred at 95°C for 12h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 650nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to obtain the trifluoroacetate salt of dark blue solid compound 2g (13.3 mg, 18.9 μmol, yield 68% ).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.52(d, J=2.8Hz, 2H), 7.26(d, J=9.7Hz, 2H), 6.95(dd, J=9.7, 2.9Hz, 2H) ,6.71(s,1H),6.69(s,1H),4.24(q,J=7.1Hz,4H),4.19(s,4H),3.88–3.79(m,16H),3.70(s,3H), 1.89(s, 3H), 1.31(t, J=7.2Hz, 6H), 0.60(s, 3H), 0.59(s, 3H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ173.25, 171.76, 155.38, 150.12, 149.88, 143.37, 140.82, 132.06, 129.96, 129.31, 122.28, 120.31, 115.58, 114.78, 6 7.59, 61.96, 56.59, 55.29, 48.36, 19.01 ,14.64,-1.30,-1.52.

HRMS (ESI) theoretical value C ₃₉ H ₅₀ N ₃ O ₇ Si ⁺ [M] ⁺ 700.3413, calculated value 700.3396.

(4) Synthesis of compound PK Zinc FarRed-2:

Compound 2g (13.3mg, 18.9μmol) was dissolved in 1mL of 12M hydrochloric acid solution, and refluxed for 30min. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, and the resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 650nm ; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) gave the trifluoroacetate salt of compound PK Zinc FarRed-2 as a dark blue solid (8.3 mg, 13 μmol, yield 68%).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.51(d, J=2.9Hz, 2H), 7.27(d, J=9.6Hz, 2H), 6.95(dd, J=9.7, 2.9Hz, 2H) ,6.75(s,1H),6.69(s,1H),4.17(s,4H),3.87–3.79(m,16H),3.72(s,3H),1.90(s,3H),0.60(s,3H ), 0.58(s,3H).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ175.41, 171.84, 155.39, 150.12, 149.87, 143.42, 140.80, 132.06, 129.98, 129.34, 122.27, 120.11, 115.59, 114.81, 6 7.59, 56.49, 55.41, 48.36, 19.03, - 1.30, -1.53.

HRMS (ESI) theoretical value C ₃₅ H ₄₂ N ₃ O ₇ Si ⁺ [M] ⁺ 644.2787, actual value 644.2761.

Example 8: Synthesis of Compound PK Zinc FarRed-3:

(1) Synthesis of compound 3d:

In 10mL of 2-methoxy-3,5-dimethylaniline (1.80g, 11.9mmol, 1.0eq) in CH ₂ Cl ₂ solution, add tetrabutyl bromide diluted with 10 mL of CH ₂ Cl ₂ dropwise over 30 min Ammonium (5.74g, 11.9mmol, 1.0eq), the resulting mixture was stirred and reacted at room temperature for 15min. After the reaction, the reaction solution was neutralized with saturated NaHCO ₃ aqueous solution, and the organic layer was extracted with CH ₂ Cl ₂ . The collected organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was purified by column chromatography (5% CHCl ₃ /Et ₂ O) to obtain pure colorless oily compound 3d (1.75 g, 7.81 mmol, yield 66%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 6.55 (s, 1H), 4.92 (s, 2H), 3.57 (s, 3H), 2.21 (s, 3H), 2.17 (s, 3H).

¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 143.10, 140.23, 132.60, 129.97, 114.49, 111.97, 58.85, 23.12, 16.39.

(2) Synthesis of compound 3e:

Compound 3d (1.13g, 4.95mmol, 1.0eq), triethylamine (1.45mL, 10.4mmol, 10.0eq) and 4-(dimethylamino)pyridine (60.0mg, 0.50mmol, 0.1eq) were dissolved at room temperature In 20mL CH ₂ Cl ₂ , then slowly drop 5mL of 1,2-bis(chlorodimethylsilyl)ethane (1.17g, 5.45mmol, 1.1eq) in CH ₂ Cl ₂ into the above mixture , stirred for 3h. After the reaction, 30 mL of n-hexane was added to precipitate triethylamine chloride salt, which was then filtered off. The obtained goose-yellow oil was purified by column chromatography (alumina substrate, 5% ethyl acetate/petroleum ether) to finally obtain compound 3e (1.03 g, 2.77 mmol, yield 56%) as a colorless oil.

¹ H NMR (400MHz, Chloroform-d) δ6.74(s,1H),3.56(s,3H),2.35(s,3H),2.30(s,3H),0.83(s,4H),0.15(s ,12H).

¹³ C NMR (101 MHz, Chloroform-d) δ 152.60, 139.36, 133.34, 132.01, 126.52, 120.55, 59.84, 23.78, 17.40, 9.19, 0.50.

(3) Synthesis of compound 3f:

Compound 3e (100mg, 0.270mmol, 4.7eq) and anhydrous THF (3mL) were added into a heat-dried flask filled with argon. After the solution was cooled to -20°C, n-BuLi (1.6M n-hexane solution 0.18mL, 0.30mmol, 5.2eq) was rapidly dropped into n-BuLi with a syringe, and the reaction was stirred at -20°C for 15min. At the same temperature, 1 mL of a solution of siloxanthone (25.0 mg, 57.8 μmol, 1.0 eq) in anhydrous THF was slowly dripped into it with a syringe, and the stirring was continued for 20 min. After the reaction was completed, 2M hydrochloric acid was added to quench the reaction and warmed to room temperature. After neutralizing the reaction solution with saturated NaHCO ₃ aqueous solution, the organic layer was extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , then the reaction solution was concentrated under vacuum/reduced pressure, and purified by HPLC (eluent, 20min linear gradient, solvent B component increased from 30% to 95%; Flow rate 5.0mL/min; detection wavelength 650nm; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give trifluoroacetic acid of dark blue solid compound 3f Salt (20.4 mg, 37.6 μmol, 66% yield).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.53 (d, J=2.8Hz, 2H), 7.20 (d, J=9.7Hz, 2H), 6.96 (dd, J=9.7, 2.9Hz, 2H) ,6.93(s,1H),3.86–3.82(m,16H),3.80(s,3H),1.91(s,3H),1.89(s,3H),0.61(s,3H),0.60(s,3H ).

¹³ C NMR (101MHz, Methanol-d ₄ )δ170.52, 155.48, 150.06, 147.55, 141.97, 136.04, 133.93, 133.11, 130.72, 129.45, 122.41, 119.23, 116.00, 67.58, 60 .81,48.42,19.48,13.38,-1.52, -1.56.

HRMS (ESI) theoretical value C ₃₂ H ₄₀ N ₃ O ₃ Si ⁺ [M] ⁺ 542.2833, actual value 542.2814.

(4) Synthesis of compound 3g:

At room temperature, K ₂ CO ₃ (50.0mg, 0.37mmol, 10.0eq), KI (3.2.mg, 19μmol, 0.5eq) and bromine were added to 2mL of compound 3f (20.0mg, 37μmol, 1.0eq) in DMF Ethyl acetate (61mg, 0.37mmol, 10.0eq), the reaction was stirred at 95°C for 12h. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 50% to 95%; flow rate 5.0mL/min; detection wavelength 650nm; elution Reagent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give dark blue solid compound 3g of trifluoroacetate (15.6 mg, 21.8 μmol, yield 59% ).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.52(d, J=2.9Hz, 2H), 7.18(d, J=9.6Hz, 2H), 6.95(dd, J=9.7, 2.9Hz, 2H) ,6.80(s,1H),4.27(s,4H),4.21(q,J=7.1Hz,4H),3.86–3.80(m,16H),3.76(s,3H),1.88(s,3H), 1.88(s, 3H), 1.28(t, J=7.1Hz, 6H), 0.60(s, 3H), 0.59(s, 3H).

¹³ C NMR (101MHz, Methanol-d ₄ )δ172.64, 171.16, 155.51, 150.05, 149.34, 143.70, 142.13, 133.46, 131.99, 131.21, 129.51, 122.35, 119.88, 115.96, 6 7.60, 61.88, 60.15, 54.55, 48.40, 19.69 ,14.60,13.46,-1.51,-1.57.

HRMS (ESI) theoretical value C ₄₀ H ₅₂ N ₃ O ₇ Si ⁺ [M] ⁺ 714.3569, actual value 715.3566.

(5) Synthesis of compound PK Zinc FarRed-3:

Compound 3g (15.6mg, 21.8μmol) was dissolved in 2mL of 12M hydrochloric acid solution, and refluxed for 30min. After the reaction, the reaction solution was concentrated under vacuum/reduced pressure, and the resulting crude product was purified by HPLC (eluent, 20min linear gradient, solvent B component rose from 30% to 95%; flow rate 5.0mL/min; detection wavelength 650nm ; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to give the trifluoroacetate salt of PK Zinc FarRed-3 as a dark blue solid (10.1 mg, 15.3 μmol, yield 68%).

¹ H NMR (400MHz, Methanol-d ₄ ) δ7.51(d, J=2.9Hz, 2H), 7.21(d, J=9.6Hz, 2H), 6.96(dd, J=9.7, 2.8Hz, 2H) ,6.80(s,1H),4.18(s,4H),3.87–3.78(m,16H),3.67(s,3H),1.86(s,6H),0.60(s,3H),0.59(s,3H ).

¹³ C NMR (101MHz, Methanol-d ₄ ) δ178.65, 171.59, 155.51, 150.00, 148.63, 143.05, 142.31, 132.53, 132.18, 131.33, 129.68, 122.25, 118.91, 116.01, 6 7.59, 60.74, 58.95, 48.39, 19.74, 13.62 ,-1.52,-1.54.

HRMS (ESI) theoretical value C ₃₆ H ₄₄ N ₃ O ₇ Si ⁺ [M] ⁺ 658.2943, actual value 658.2947.

Synthesis of Example 9 Compound PK SiR Morpho:

Add compound 1h (76mg, 0.270mmol, 4.7eq) and anhydrous THF (3mL) in a heat-dried flask filled with argon. After the solution was cooled to -20°C, n-BuLi (1.6M n-hexane solution 0.18mL, 0.30mmol, 5.2eq) was rapidly dropped into n-BuLi with a syringe, and the reaction was stirred at -100°C for 15min. At the same temperature, 1 mL of a solution of siloxanthone (25.0 mg, 57.8 μmol, 1.0 eq) in anhydrous THF was slowly dripped into it with a syringe, and the stirring was continued for 20 min. After the reaction was completed, 2M hydrochloric acid was added to quench the reaction and warmed to room temperature. After neutralizing the reaction solution with saturated NaHCO ₃ aqueous solution, the organic layer was extracted with EtOAc. The organic layer was washed with brine, dried over Na ₂ SO ₄ , then the reaction solution was concentrated under vacuum/reduced pressure, and purified by HPLC (eluent, 20min linear gradient, solvent B component increased from 30% to 95%; Flow rate 5.0mL/min; detection wavelength 650nm; eluent A: ddH ₂ O containing 0.1% (v/v) TFA; eluent B: CH ₃ CN) to obtain dark blue solid compound PK SiR Morpho trifluoro Acetate (23.0 mg, 39.9 μmol, 70% yield).

¹ H NMR (400MHz, Methanol-d ₄ )δ7.88(s, 2H), 7.57(d, J=2.8Hz, 2H), 7.13(d, J=9.6Hz, 2H), 6.97(dd, J= 9.6, 2.7Hz, 2H), 3.89–3.83(m, 16H), 2.06(s, 6H).

Example 10 Zinc ion titration and dissociation constant (K _d ) test method of zinc ion probes PK Zinc Red 1-5 and PK Zinc FarRed 1-3.

(1) Preparation of test solutions: Various test solutions containing 1 μM PK Zinc dye were prepared by mutual dilution of "zero zinc ion buffer" and "high concentration zinc ion buffer". The zero zinc ion solution that needs to be prepared first contains 100mM pH 7.4HEPES, 100mM NaNO ₃ ; the "high concentration zinc ion buffer" contains 100mM pH 7.4HEPES, 100mM NaNO ₃ and 10mM ZnSO ₄ . For a buffer system with a free zinc ion concentration greater than 100 nM, directly use two buffers for mutual dilution. For the buffer system with free zinc ion concentration less than 100nM, add 10mM NTA to chelate high concentration zinc ion again in the solution, and by literature (Org.Lett.,2011,13(17):4558-4561) The formula calculates the actual free zinc ion concentration in the buffer system.

(2) Spectrum test: The fluorescence spectrum was tested on Shimadzu RF-5301PC at room temperature, and the absorption spectrum was tested on UV-1780 at room temperature. The fluorescence spectra and absorption spectra of all zinc ion dyes described in the present invention were repeated three times at each zinc ion concentration using separately prepared solutions.

The fluorescence spectrum test results of different dyes at different zinc ion concentrations are shown in Figures 1, 3, 5, 7, 9, 11, 13, and 15 (the inset figure in the upper right corner is the absorption spectrum), and the concentration titration test results are shown in Figure 2 , 4, 6, 8, 10, 12, 14, 16. K _d is calculated according to the equation F _m (FminKD+Fmax[Zn ²⁺ ])/(Kd+[Zn ²⁺ ]). Wherein F is the fluorescence intensity at a given zinc ion concentration [Zn ²⁺ ], F _min is the fluorescence intensity when [Zn ²⁺ ]=0; F _max is the fluorescence intensity when the dye is saturated with zinc ions. Important photophysical properties of the compounds PK Zinc Red 1-5 and PK Zinc FarRed 1-3 according to the invention are listed below Table 1:

Table 1

Example 11 A test method for observing the co-release of insulin/zinc ions on pancreatic beta cells or isolated pancreatic islet tissue using zinc ion probes.

Islets were cultured on 35mm glass-bottom confocal dishes (Cellvis, D35-14-1-N) for 24 hours, then washed twice and incubated with 125mM NaCl, 5.9mM KCl, 2.4mM _CaCl2 , 1.2mM _MgCl2 , 1mM Preheat for about 15 minutes in a KRBB solution of L-glutamine, 25 mM HEPES, 3 mM glucose, 0.1% bovine serum albumin, 10% glucose and 1 μM zinc ion dye. Next, islets were stimulated with KRBB solutions containing indicated glucose concentrations or/and drugs and imaged. Fluorescence images17,18,19,20 were all acquired with an Olympus inverted IX-81 laser confocal microscope mounted on a CSU-X1 turntable. The image is collected by 60X (NA1.35, Olympus) or 100X (NA1.30, Olympus) oil lens, the sampling rate of monochrome is ~2Hz, the sampling rate of dual channel is ~1Hz, and the sampling rate of four-color imaging is ~0.3Hz.

Example 12 Test method for immunofluorescence imaging using PK SiR Morpho and commercial dyes Alexa 647 and Cy5.

Co-incubate the dye and secondary antibody in PBS 7.4 buffer at a concentration ratio of 20:1, use Sephadex G-25 column or desalting column to separate the dye-labeled secondary antibody and free dye and use Nanodrop 2000 determines the average number of dye-labeled secondary antibodies by A ₂₆₀ /A ₆₅₀ . Immunofluorescent staining was then performed on fixed HeLa cells and imaged on a high-content live-cell imaging system. Fluorescent signal is recorded by short-term shooting + 1s 650nm laser irradiation, and each cycle is counted once. The specific shooting time and laser light intensity are: PK SiR Morpho 100ms, 50%; Alexa 64750ms, 50%; Cy520ms, 50% . The curve of the recorded fluorescence signal changing with time is shown in FIG. 21 .

Claims (10)

1. A class of rhodamine fluorescent dyes containing water-soluble substituents, characterized in that the series of dyes has the following general structural formula: Wherein R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ are independent of each other; R ₁ is O or Si(CH ₃ ) ₂ ; _R2 is O; _R3 is _CH2 ; R ₄ is H or CH ₃ respectively; R ₅ is H, OCH ₃ , OCH ₂ CH ₃ , OCH ₂ CH ₂ OCH ₃ , OCH ₂ COOH or R ₆ is COOH or N(CH ₂ COOH) ₂ ; _R7 is H; R ₈ are H or CH ₃ respectively; X- is Cl ^- or CF ₃ COO ^- . 2. The fluorescent dye according to claim 1, wherein said R ₁ is O, R ₂ is O, R ₃ is CH ₂ , R ₄ is H, R ₅ is OCH ₃ , R ₆ is N (CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, X- is CF ₃ COO ^- . 3. The fluorescent dye according to claim 1, characterized in that, said R ₁ is O, R ₂ is O, R ₃ is CH ₂ , R ₄ is H, R ₅ is OCH ₂ CH ₃ , R ₆ N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, X- is CF ₃ COO ^- . 4. The fluorescent dye according to claim 1, characterized in that, said R ₁ is O, R ₂ is O, R ₃ is CH ₂ , R ₄ is H, R ₅ is OCH ₂ CH ₂ OCH ₃ , R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, and X- is CF ₃ COO ^- . 5. The fluorescent dye according to claim 1, characterized in that, said R ₁ is O, R ₂ is O, R ₃ is CH ₂ , R ₄ is H, R ₅ is OCH ₂ COOH, R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, X- is CF ₃ COO ^- . 6. The fluorescent dye according to claim 1, characterized in that, said R ₁ is O, R ₂ is O, R ₃ is CH ₂ , R ₄ is H, R ₅ is R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, and X- is CF ₃ COO ^- . 7. The fluorescent dye according to claim 1, characterized in that, said R ₁ is Si(CH ₃ ) ₂ , R ₂ is O, R ₃ is CH ₂ , R ₄ is H, and R ₅ is OCH ₃ , R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, X- is CF ₃ COO ^- . 8. The fluorescent dye according to claim 1, characterized in that, said R ₁ is Si(CH ₃ ) ₂ , R ₂ is O, R ₃ is CH ₂ , R ₄ is CH ₃ , R ₅ is OCH ₃ , R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is H, X- is CF ₃ COO ^- . 9. The fluorescent dye according to claim 1, characterized in that, said R ₁ is Si(CH ₃ ) ₂ , R ₂ is O, R ₃ is CH ₂ , R ₄ is CH ₃ , R ₅ is OCH ₃ , R ₆ is N(CH ₂ COOH) ₂ , R ₇ is H, R ₈ is CH _{3 ,} X- is CF ₃ COO ^- . 10. The fluorescent dye according to claim 1, characterized in that, said R ₁ is Si(CH ₃ ) ₂ , R ₂ is O, R ₃ is CH ₂ , R ₄ is CH ₃ , R ₅ is H , R ₆ is COOH, R ₇ is H, R ₈ is CH ₃ , X ^- is Cl ^- .